ABSTRACT This project aims at elucidating the mechanisms underlying epigenetic regulation of hepatitis B virus (HBV) covalently closed circular (ccc) DNA transcription in hepatocytes, focusing on viral X protein (HBx)- mediated epigenetic regulation of HBV cccDNA with involvement and fine balancing of host epigenetic modulators. HBV cccDNA is essential to the virus life cycle, its complete elimination or inactivation during chronic infection is considered critical to a cure but has not been achieved by current antivirals. HBV cccDNA exists in the cell nucleus as an individual minichromosome decorated with histones and non-histone proteins. Elucidating the mechanisms of chromatin compactization of cccDNA and principles of epigenetic regulation of cccDNA episome in its interplay with host factors could allow us to elaborate new antiviral strategies for addressing the unmet clinical need. Among the limited number of HBV-encoded proteins, the viral regulatory protein HBx serves as a multifunctional transactivator of the viral and cellular promoters and has been proven to be a potent epigenetic modifying factor in HBV-infected livers. To further address the role of HBx in cccDNA transcription, we have developed a pair of inducible cccDNA reporter stable cell lines with and without HBx expression, namely HepBHAe82 and HepBHAe∆x67. While both cell lines are able to produce comparable level of cccDNA regardless of the presence or absence of HBx, the cccDNA in HepBHAe∆x67 cells is epigenetically silenced. The HBx-dependent cccDNA transcription has also been recapitulated in wildtype and HBx-minus HBV infected hepatocytes. In this project, by making use of these experimental systems, we will systematically characterize the epigenetic profile variations between transcriptionally active and inactive cccDNA (Aim 1), map the interaction of cccDNA minichromosome with host chromosomes (Aim 2), and identify host epigenetic modulators that regulate cccDNA transcription through comparative proteomic approach, followed by functional validation (Aim 3). In Aim 3, we have already identified HMGB1 as a novel host restriction factor for cccDNA, and will further elucidate the mechanism of HMGB1-mediated epigenetic repression of cccDNA transcription and the interplay between HBx and HMGB1 in cccDNA activation. The accomplishment of this project will shed more light on the cccDNA epigenetics, and provide novel antiviral targets for development of therapeutics that epigenetically silence cccDNA to achieve a functional cure of chronic hepatitis B.